Tissue Engineering: 4 Aspects (With Diagram)

This article throws light upon the four fundamental and basic aspects of tissue engineering.

The four fundamental aspects are: (1) Cell Sources and Culture (2) Cell Orientation (3) Cell Support Materials and (4) Design and Engineering of Tissues.

Tissue engineering (TE) refers to the application of the principles of engineering to cell culture for the construction of functional anatomical units (tissues/organs). The ultimate purpose of TE is to supply various body parts for the repair or replacement of damaged tissues or organs.

Tissue engineering may be regarded as the backbone of reconstructive surgery. It is possible to supply almost all surgical implants (skin, blood vessels, ligaments, heart valves, joint surfaces, nerves) through the developments in tissue engineering.

There are two schools of thought while dealing with tissue engineering techniques:

1. Some workers believe that the living cells possess an innate potential of biological regeneration. This implies that when suitable cells are allowed to grow on an appropriate support matrix, the cells proliferate, and ultimately result in an organized and functional tissue. This tissue resembles the original tissue in structure and function. This approach is very simple, and economical, although the success is limited.

2. According the second school of thought, there are several control processes to produce a new and functional tissue. Thus, tissue regeneration in vivo or tissue production in vitro are very complex. Therefore, tissue engineering is not a simple regeneration of cells, and it requires a comprehensive approach with a thorough understanding of cellular configuration, special arrangement and control process.

Tissue engineering is a complicated process. Some fundamental and basic aspects of TE with special reference to the following aspects are briefly described:

1. Cell sources and culture

2. Cell orientation

3. Cell support materials

4. Design and engineering of tissues.

1. Cell Sources and Culture:

Adequate quantities of cells are required for tissue engineering. There are three types of cell sources-autologous, allogeneic and xenogeneic.

Autologous Cell Sources:

The cell source is said to be autologous when the patient’s own cells are used in TE. This is a straight forward approach. A piece of desired tissue is taken by biopsy. It may be enzymatically digested or explant cultured, and the cells are grown to the required number.

The main advantages of autologous cells in TE are:

i. Avoidance of immune complications

ii. Reduction in the possible transfer of inherent infections.

There are certain disadvantages associated with autologous cells.

i. It is not always possible to obtain sufficient biopsy material from the patient.

ii. Disease state and age of the patient will be limiting factors.

Allogeneic Cell Sources:

If the cells are taken from a person other than the patient, the source is said to be allogeneic.

The advantages of allogeneic cell source are listed:

i. Obtained in good quantity from a healthy donor.

ii. Cells can be cultured in a large scale.

iii. Cost-effective with consistent quality.

iv. Available as and when required by a patient.

The major problem of allogeneic cell source is the immunological complications that may ultimately lead to graft rejection. The immune responses however, are variable depending on the type of cells used. For instance, endothelial cells are more immunogenic while fibroblasts and smooth muscle cells are less immunogenic. The age of the donor is another important factor that contributes to immunological complications. Thus, cells from adult donors are highly immunogenic while fetal or neonatal cells elicit little or no immune response.

Xenogeneic Cell Sources:

When the cells are taken from different species (e.g. pig source for humans) the source is said to be xenogeneic. This approach is not in common use due to immulogical complications.

Culture of Cells:

The methods adapted for culturing of cells required for tissue engineering depend on the type and functions of cells. For most of the cells, the conventional monolayer cultures serve the purpose. The major drawback of monolayer cultures is that cells may lose their morphology, functions and proliferative capacity after several generations. Some workers prefer three dimensional cultures for the cells to be used in tissue engineering. The nutrient and gaseous exchanges are the limiting factors in three dimensional cultures.

Genetic Alterations of Cultured Cells for Use in TE:

Gene therapy can be successfully employed in tissue engineering. This can be achieved by transferring the desired genes to cells in culture. The new genes may increase the production of an existing protein or may synthesize a new protein.

Some success has been achieved in this direction:

i. Genetically altered fibroblasts can produce transferrin, clotting factor VIII and clotting factor IX.

ii. Modified endothelial cells can synthesize tissue plasminogen activator.

iii. Genetically engineered keratinocytes can produce trans-glutaminase-l (This enzyme is lacking in patients suffering from a dermal disorder, lamellar icthyosis). The altered keratinocytes proved successful when transplanted in animal (rat) models of this disease.

2. Cell Orientation:

The orientation of cells with regard to specific shape and spatial arrangement is influenced by the following environmental factors:

1. Substrate or contact guidance.

2. Chemical gradients.

3. Mechanical cues.

Substrate Guidance:

The topographical features of the substrate determine the contact guidance. These features may be in the form of ridges, aligned fibers etc. It is possible to use differential attachment to substrates as a means of producing different alignment of cells. In recent years, synthetic polymer substrate collagen fibrils and fibronectin are used as bioresorbable templates for tissue engineering.

Chemical Gradients:

Development of chemical gradients is required for cellular orientation and for the stimulation of cellular functions. Certain growth factors and extracellular macromolecules are capable of creating chemical gradients e.g. vascular endothelial growth factor (VEGF), oligosaccharide fragments of hyaluronan, fibronectin, and collagen. There are certain practical difficulties in maintaining effective chemical gradients for the cells in three dimensional cultures. This is particularly the limiting factor when the cells become dense.

Mechanical Cues:

The response of the cells to mechanical signals is complex and this may result in any one or more of the following:

i. Changes in the cell alignment.

ii. Deformation of cytoskeleton.

iii. Altered matrix formation.

iv. Synthesis of regulatory molecules (e.g growth factors, hormones).

There are mainly three mechanical cues governing cell populations:

1. Tensional forces.

2. Compressional forces.

3. Shear forces.

3. Cell Support Materials:

The support materials of cells largely determine the nature of adherent cells or cell types, and consequently tissue engineering.

There are a large number of support materials which may be broadly categorized as follows:

i. Traditional abiotic materials.

ii. Bio-prosthesis materials.

iii. Synthetic material.

iv. Natural polymers.

v. Semi-natural materials.

Traditional Abiotic Materials:

The traditional abiotic support materials include plastics, ceramics and metals. These materials cannot be resorbed or become biologically integrated into the tissues. Therefore, it is preferable to avoid the traditional materials in tissue engineering.

Bio-prosthesis Materials:

The natural materials modified to become biologically inert represent bio-prosthesis materials. They are formed by extensive chemical cross linking of natural tissues. For instance, the natural collagen-based connective tissue (e.g. porcine heart values) can be stabilized by treatment with glutaraldehyde.

The product formed is non-immunogenic that remains unchanged at the site of transplantation for several years. However, growth of some cells or even connective tissue can occur on bio-prosthesis materials. The design and fabrication of these materials is done in such a way that their functions are not affected by the surrounding host tissues.

Synthetic Materials:

A wide range of synthetic bioresorbable polymers are available as support materials. The most commonly used polymers in tissue engineering are poly (glycolic acid) (PGA), poly (lactic acid) (PLA), and copolymer PLGA (poly (lactic-co-glycolic acid). The composition and dimensions of these polymers can be so adjusted to make them stable in vivo, besides supporting in vivo cell growth.

There are certain advantages in using synthetic polymers:

i. Production is easy and relatively cheap.

ii. Composition of polymers is reproducible even in large scale production.

There are however, some disadvantages also:

i. Compatibility with cells is not as good as natural polymers.

ii. On degradation, they may form some products which cause undesirable cellular effects.

Natural Polymers:

The most widely used natural polymer materials are collagen-chondroitin sulfate aggregates. These materials are commercially available with varying composition under the trade name Integra. The other natural polymers for cell support are usually obtained by their aggregation in culture as it occurs in vivo e.g. collagen gels, fibrin glue, Matrigel and some polysaccharides.

Among the polysaccharides, chitosan and hyaluronan are used as hydrated gels. The natural polymers mainly act on the principle of intermolecular interaction within the polymers to promote intimate molecular packing. The so formed molecules can effectively serve as support materials.

Semi-natural Materials:

Semi-natural materials are derived from the natural macromolecular polymers or whole tissues. They are the modified materials to achieve aggregation or stabilization.

Some examples of semi-natural materials are listed below:

i. Chemically cross-linked hyaluronan, stabilized by benzyl esterification.

ii. Collagen cross-linked with agents such as tannic acid or carbodiimide.

4. Design and Engineering of Tissues:

The following surgical criteria are taken into consideration while dealing with tissue engineering:

i. Rapid restoration of the desired function.

ii. Ease of fixing the tissue.

iii. Minimal patient discomfort.

For designing tissue engineering, the source of donor cells is very critical. Use of patients own cells (autologous cells) is favoured to avoid immunological complications. Allogeneic cells are also used, particularly when the TE construct is designed for temporary repair. It is observed that when the cells are cultured and/or preserved (i.e. cryopreservation), the antigenicity of allogeneic cells is reduced.

Another important criteria in TE is the support material, its degradation products, cell adhesion characteristics and mechanical cues. The design and tissue engineering with respect to skin, urothelium and peripheral nerve are briefly described hereunder.

Tissue Engineered Skin:

It was first demonstrated in 1975 that human keratinocytes could be grown in the laboratory in a form suitable for grafting. Many improvements have been made since then. It is now possible to grow epithelial cells to produce a continuous sheet which progresses to form carnified layers.

The major difficulty with TE skin is the dermal layer possessing blood capillaries, nerves, sweat glands and other accessory organs. Some developments have occurred in recent years to produce implantable skin substitutes which may be regarded as tissue engineering skin constructs.

Integra™:

This is a bio artificial material composed of collagen-glycosaminoglycan. Integra™ is not a true TE construct. It is mainly used to carry the seeded cells.

Dermagraft™:

This is composed of poly (glycolic acid) polymer mesh seeded with human dermal fibroblasts from neonatal foreskins.

Apligraf™:

This has human dermal fibroblasts seeded into collagen gel. A layer of human keratinocytes is then placed on the upper surface. The tissue constructs described above have limited shelf-life (about 5 days). However, they can integrate into the surrounding normal tissue and form a good skin cover. Further, there is no evidence of immunological complications with TE constructs.

Tissue Engineered Urothelium:

It is now possible to culture urothelial cells and bladder smooth muscle cells. This raises the hope that the construction of TE urothelium is possible. In fact, some success has been reported in the development of a functional bladder in dogs.

For this purpose, poly (glycolic acid) polymer base was shaped into a bladder and muscle cells were coated on the outer surface. The lumenal surface (i.e. inner surface) coated with pre-cultured urothelial cells. The bladder constructed in this way functioned almost like a normal one, and was maintained for about one year.

Tissue Engineered Peripheral Nerve Implants:

Peripheral nerve injury is a common occurrence of trauma and tumor resection surgery, often leading to irreversible muscle atrophy. Therefore, the repair of injured peripheral nerves assumes significance.

A diagrammatic representation of the basic design of a peripheral nerve implant is depicted in Fig 40.4.

The regeneration of the injured nerve occurs from the proximal stump to rejoin at distal stump. The regeneration is guided by three types of substances.

Conduct material:

This is the outer layer and is the primary source of guidance. Conduct material is composed of collagen-glycosaminoglycan’s, PLGA (poly lactic-co-glycolic acid), hyaluronan and fibronectin. All these are bioresorbable materials.

Filling material:

This supports the neural cells for regeneration, besides guiding the process of regeneration. Filling material contains collagen, fibrin, fibronectin and agarose.

Additives:

Additives include a large number of growth factors; neurotrophic factors (in different forms, combinations, ratios) e.g. fibroblast growth factor (FGF), nerve growth factor (NGF). Additions of Schwann cells or transfected fibroblasts promote nerve generation process.

Tissue Modeling:

Research is in progress to create tissue models in the form of artificial organs. Some of the recent development on experimental tissue modeling are briefly outlined.

Artificial liver:

Hepatocytes, cultured as spheroids or hepatocytes and fibroblasts cultured as hetero­spheroids can be used. They are held in the artificial support systems such as porous gelatin sponges, agarose or collagen. Addition of exogenous molecules is useful for the long – term culture of liver cells. Some progress has been reported in creating artificial liver as is evident from the hepatocytes three-dimensional structure and metabolic functions.

Artificial pancreas:

Spheroids of insulin secreting cells have been developed from mouse insulinoma beta cells. Some workers could implant fetal islet-like cell clusters under the kidneys of mice, although the functions were not encouraging due to limitation of oxygen supply.

Other Tissue Models:

Pituitary gland:

Multicellular spheroids could be created to study certain hormonal release e.g. luteinizing hormone (LH), following stimulation by luteinizing hormone releasing hormone (LHRH). Some success has also been achieved to create spheroids for the production of melatonin.

Thyroid gland:

Thyroid cell spheroids can be used for the study of cell adhesion, motility, and thyroid follicle biogenesis.

Brain cell cultures:

Three dimensional brain cell cultures have been used for the study of neural myelination and demyelination, neuronal regeneration, and neurotoxicity of lead. Aggregated brain cells are also used for the study of Alzheimer’s disease and Parkinson’s disease.

Heart cell cultures:

Aggregated heart cells have been used for the study of cardiac development and physiology.